Introduction

Glaucoma is the leading cause of irreversible blindness worldwide. This disease consists of the development of a chronic progressive optic neuropathy characterized by increased cupping of the optic disc, associated with progressive vision loss. This disease is related to multiple risk factors such as increased intraocular pressure; diagnosis of arterial hypertension and diabetes, ethnicity; and age ¹. It is estimated that there are currently more than 67 million patients affected by glaucoma, estimating a worldwide prevalence of over 15% for Afro-descendant communities, of which 10% present bilateral blindness ^2,3. One of the most complex aspects of this disease is the gradual development of its symptoms. Vision loss is progressive and irreversible, and visual field loss is usually only recognized by the patient in advanced stages. Although the determination of intraocular pressure is the most appropriate screening method in cost-effective terms for the identification of suspected cases of glaucoma, it is not enough to establish the diagnosis of the disease. There must be evidenced at least one other clinical criterion that demonstrates damage of the optic nerve, such as increased cupping of the optic nerve, visual field loss, or the detection of hemorrhages at the level of the optic disc ⁴.

In developed countries, it is estimated that more than 50% of people with glaucoma have not been diagnosed; in developing countries, this lack of diagnosis could be even higher, in the order of 60 to 80% ^5,6. It is suggested that up to 90% of cases of blindness due to glaucoma could be avoided through early detection and timely pharmacological treatment ⁷.

In previous studies in six Colombian cities where the prevalence of glaucoma was estimated in patients with hypertension and diabetes ^8,9, a Raizal family with a history of visual diseases was identified in the archipelago of San Andrés and Providencia, where some of their members were diagnosed with glaucoma. This suggests a possible hereditary genetic factor related to the development of this type of pathology in this family group.

In the present investigation, a whole exome analysis was carried out to identify potentially harmful non-synonymous single nucleotide molecular variants in genes associated with glaucoma pathology in members of a Raizal family from San Andrés and Providencia with a history of presenting visual diseases.

Materials and Methods

Results

Figure 1 represents the family relationships among the members of the Raizal family and the study participants who underwent exome analysis. Four of the seven participants belonged to the third generation and three to the fourth, according to the heredogram constructed from the information collected by interviewing family members. Of the four participating individuals of generation III, two groups of siblings were identified: participating individuals III-5 and III-7, half-siblings of the same father but different mother; and participating individuals III-14 and III-15. Except for III-4, all were diagnosed with glaucoma. The parents of these two groups were identified as siblings; and both had been diagnosed with glaucoma: individuals II-2 and II-4.

Figure 1 Heredogram of a Raizal family members from San Andres, Providencia, and Santa Catalina with a history of glaucoma. (*) RE= right eye; LE= left eye. Normal visual acuity is equal to 20/20. IOP = intraocular pressure. Red circles identify study participants. Two non-family-members Raizal participants (g26F and g27F) with no diagnosed visual diseases were included in the analysis to identify possibly pathogenic molecular variants. The last column shows the glaucoma diagnoses (as positive or negative ones). 

Regarding the IV generation, the participating individuals IV-5 and IV-6 were brothers, sons of III-7. Of this group of siblings, IV-5 was suspected of (presenting / suffering glaucoma. In this same generation participated individual IV-9, diagnosed with glaucoma, whose mother, individual IV-13, had also been diagnosed with glaucoma. IV-5 was the niece of III-14 and III-15 (Figure 1).

Figure 2 shows the percentage estimate of the ancestral genetic components of the study participants. In the principal component’s analysis, it was found the separation of the three ancestral populations: the separation of the African from the European and East Asian with a PC1 equal to 37.8%, and the separation of the European from the African and East Asian with a PC2 equal to 24.8%.

Figure 2 Ancestral genetic components of Raizal individuals from San Andres, Providencia and Santa Catalina. The estimate was made from the analysis of 250 informative markers of ancestry. Each participating individual was compared with data from 1,305 subjects from three distinct continental populations: African (AFR), European (EUR), and East Asian (EAS), available in the 1,000 Genomes project database. N.F.= Not familiar. 

Six of nine Raizal people presented a mixture of ancestral genetic components of African, European and East Asian origin; two with African and European origin; and one with African and East Asian genetic ancestry. However, the predominant ancestral genetic component was African, with a percentage greater than 50%, except for one participant who did not belong to the Raizal family, N.F_g26F, who presented an ancestral component mainly from East Asia, with a percentage greater than 50% (Figure 2).

Table 1 shows the number of non-synonymous single nucleotide variants and InDels identified in the exomes of study participants. The average number of non-synonymous single nucleotide variants identified in the members of the Raizal family was 12,366 ±190; and in the two Raizal participants not related to the family, it was 11,772 ±512. In relation to the InDels, in the members of the Raizal family, it was 241 ± 31; and in the two non-family related Raizal participants, it was 210 ±2. The mean number of non-synonymous single nucleotide variants and InDels that have already been reported were 10,053 and 185, respectively. The number of reported non-synonymous single nucleotide variants shared by family members were 33.88; and InDels 68. When comparing the molecular variants of the members of the family vs. non-members, it was found that they didn’t share 423 non-synonymous single nucleotide variants, nor 11 InDels. These possibly pathogenic molecular variants were distributed in 197 genes. By filtering the genes with the list available in DisGeNET, six genes associated with glaucoma were identified.

Table 2 shows the genes identified with their single nucleotide variant molecular variants that are present in family members, compared with non-members. The identified genes were: BTC (betacellulin); CENPJ (Centromere J protein); DHDDS (dehydrodolichol diphosphate synthase); TFR (Transferrin Receptor); and WLS (receptor for Wnt proteins on secretory cells).

Table 3 presents the damage prediction of each identified non-synonymous single nucleotide variant. The WLS gene presented the non-synonymous single nucleotide variant rs983034 with a prediction of high damage to protein structure and function. The rs983034 was present in all members of the Raizal family; but it was absent in non-family members (Table 2). The TFRC gene presented the non-synonymous single nucleotide variant rs3817672 with a prediction of great damage in six of the members of the Raizal family. In the CENPJ gene, rs7336216 was identified with high damage prediction in four family members. Finally, the BTC gene presented the non-synonymous single nucleotide variant rs11938093 with high damage prediction in four of the family members. The DHDDS gene did not present non-synonymous single nucleotide variants with predicted harmfulness.

Discussion

Complete exome analysis of a Raizal family group from the archipelago of San Andres and Providencia, Colombia, was performed in the present study. Several members of this family had been diagnosed with glaucoma, and it was possible to identify four harmful non-synonymous single nucleotide variants in genes associated with the disease: rs11938093 in the BTC gene; rs7336216 in the CENPJ gene; rs3817672 in the TFRC gene; and rs983034 in the WLS gene. Interestingly, single nucleotide variant rs983034 in the WLS gene was identified only in members of the Raizal family. Likewise, the non-synonymous single nucleotide variants rs3816539 in the DHDDS gene and rs28549760 in the BTC gene were identified only in family members but with low damage prediction. Based on the heredogram that was constructed with the information obtained from the participants and the clinical history of those diagnosed with glaucoma (Figure 1), it can be suggested that the non-synonymous single nucleotide variants rs983034, rs3816539 and rs28549760 present autosomal dominant segregation, just like the disease. This coincides with Meneses et al., 2011 ²⁷, who reported that 43.1% of glaucoma cases are familial; these, 56.3% show an autosomal dominant inheritance pattern, 38.9% an autosomal recessive form, and 2.8% do not present a defined pattern.

According to the clinical history records, the relatives with glaucoma from levels I to III in the hederogram were diagnosed after the age of 55 years, while the three relatives with glaucoma, and the one with suspected level IV glaucoma were diagnosed before the age of 47 years. Due to the above, it is possible that (they / some of them) are presenting a type of juvenile glaucoma caused by low-frequency variants; however, it is necessary to increase the number of samples to make future analyses adjusted for age, as well as for other risk factors, such as systemic arterial hypertension and diabetes mellitus ^8,9.

It was also possible to determine that African is the main ancestral genetic component of the group of Raizal participants from the archipelago of San Andres and Providencia in the Colombian Caribbean; followed by the European, possibly as a consequence of the arrival of these human groups to the Americas due to the trafficking of enslaved people and ethnic mixture between the 16th and 19th centuries, due to the expansion of the British empire; and later, the Spanish one ^28,29. Likewise, ancestral components of East Asia were found, being in one person the main one, possibly due to the arrival of people at the archipelago from China, between 1839 and 1917, in search of work in the growing fields ³⁰. Hence, the expanded denomination of the term Raizal is both for people with African genetic ancestry and Chinese in the archipelago of San Andres and Providencia. The present investigation confirmed that the Raizal family under study had an African genetic ancestral component (Figure 2). Furthermore, studies conducted in African-Americans have indicated that visual impairment, vision loss, and the diagnosis of angle glaucoma are more frequent in this population than in Europeans / European descendants ^31,32.

On the genes identified with non-synonymous single nucleotide variant with the prediction of high protein damage, rs983034 was identified in the WLS gene in all members of the Raizal family (Tables 2 and 3). The WLS gene encodes a receptor for Wnt proteins in secretory cells that regulates this signaling pathway. The Wnt signaling pathway is involved in highly conserved signal transduction during development that regulates various cellular functions ^33,34. In eye development, Wnt signaling controls multiple developmental processes and morphogenic patterns. These processes include the dorsoventral pattern of the optic cup, the lens, the retinal pigment epithelium, the vascular system, and the ciliary margin ^35-39. Likewise, Wnt signaling has been widely related to ocular diseases such as retinal degeneration, cataracts, congenital ocular dysfunctions ⁴⁰ and glaucoma ^41,42.

The main risk factor for glaucoma, especially for primary open-angle glaucoma, is increased intraocular pressure. Elevated intraocular pressure in patients with glaucoma is due to glaucomatous lesions of the trabecular meshwork and impaired function of the latter, with increased resistance to aqueous humor outflow due to excessive deposition of extracellular matrix proteins. A study showed that the WLS-regulated Wnt cell signaling pathway controls homeostasis of the trabecular meshwork in a spatiotemporal manner; and its inhibition is associated with an increase in intraocular pressure, which leads to the pathology of glaucoma; on the contrary, its activation after inhibition reverses the pathological phenotype ⁴³.

The non-synonymous single nucleotide variant rs3817672 in the TFRC gene with high damage prediction was identified in six of the seven family members. The family member in whom this single nucleotide variant was not identified had a negative diagnosis for glaucoma. The TFRC gene encodes a cell surface receptor required for cellular iron uptake through the process of receptor-mediated endocytosis. The degradation of TFCR is induced by the overexpression of the protein Optineurin (OPTN) by a mechanism that is still unknown ⁴⁴, and the recruitment of the RAB12 protein, leads to the death of retinal ganglion cells by autophagy ⁴⁵, which could ultimately lead to the pathology of glaucoma.

Two non-synonymous single nucleotide variants were identified in the BTC gene, where one of them was identified in four members of the family, rs11938093, with a prediction of high damage. The BTC gene encodes a protein part of the epidermal growth factors family. BTC plays a crucial role in the regulation of retinal vascular permeability ⁴⁶. A study showed that glycosylation of this protein has proliferative effects on retinal pigment epithelial cells, suggesting that it is closely related to retinal vascular etiology and pathogenesis ⁴⁷. Furthermore, a study conducted in a mouse animal model with glaucoma showed that BTN messenger RNAs are inhibited by microRNA-149, which was related to increased apoptosis of murine retinal ganglion cells ⁴⁸.

The non-synonymous single nucleotide variant rs7336216 predicting high damage in the CENPJ gene was identified in four of the seven members of the raizal family. CENPJ encodes a protein that belongs to the family of centromere proteins, which, during cell division, play an important role in maintaining the integrity of the centrosome. Likewise, it has been described that the CENPJ protein can function as a transcriptional coactivator in the Stat5 signaling pathway, and NF-kappa B. single nucleotide variant in this gene have been related to diseases such as primary autosomal recessive microcephaly and Seckel syndrome ⁴⁹. Seckel syndrome is associated with ocular defects in humans, including spontaneous lens dislocation, myopia, astigmatism, and retinal degeneration ⁵⁰. Low expression of CENPJ has been detected in the retinal neuroblast layer of mice that have exhibited a number of ocular abnormalities compared to healthy mice ⁵¹.

Regarding the DHDDS gene, a non-synonymous single nucleotide variant was identified in all members of the Raizal family, rs3816539; however, the prediction of damage was low. The DHDSS gene encodes part of the enzymatic complex dehydrodolichol diphosphate synthase, an enzyme that participates in the synthesis of Dolichol monophosphate at the retina level; dolichol acts as an "anchor" at the level of the membrane of the endoplasmic reticulum of the rods of the eye, allowing the glycosylation of synthesized rhodopsin. The activity of DHDDS must be balanced since the lack of glycosylation of rhodopsin affects both the function of this protein and its intracellular traffic, leading to the progressive degeneration of photoreceptors ⁵².

Conclusion

In the present study, four non-synonymous single nucleotide variant molecular variants were identified with a prediction of high damage to gene protein structure and function associated with glaucoma pathology in an Afro-Colombian Raizal family with a history of this disease. Interestingly, single nucleotide variant rs983034 in the WLS gene was identified only in members of the Raizal family. Likewise, the non-synonymous single nucleotide variants rs3816539 in the DHDDS gene and rs28549760 in the BTC gene were identified only in family members, but with low damage prediction. Despite the above, we do not rule out the possibility that these last two single nucleotide variants can help screen for the risk opportunity of developing glaucoma in this Raizal family; and in the population with African ancestral components in general.

Due to the small size of the control group sample, there is a limitation in identifying the disease-causing variants in these patients. Thus, given the high frequency in different populations of the selected variants, there is a high probability that these variants were chosen at random because they were not represented in the two control subjects used in the study. Additional studies will be needed to test this hypothesis.